Computational Reconstruction of Multidomain Proteins Using Atomic Force Microscopy Data

SummaryClassical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally based test cases: Tobacco mosaic virus, a rod-shaped virus; and Aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multicomponent structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures

Laue diffraction protein crystallography at the National Synchrotron Light Source

A new facility for the study of protein crystal structure using Laue diffraction has been established at the X26 beam line of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory. The characteristics of the beam line and diffraction apparatus are described. Selected results of some of the initial experiments are discussed briefly by beam line users to illustrate the scope of the experimental program. Because the Laue method permits the recording of large data sets in a single shot, one goal in establishing this facility has been to develop the means to study time-resolved structures within protein crystals. Systems being studied include: the reactions catalyzed by trypsin; photolysis of carbonmonoxy myoglobin; and the photocycle of photoactive yellow protein

Recognition and interactions controlling the assemblies of beta barrel domains.

We present a qualitative computer graphics approach to the characterization of forces important to the assembly of beta domains that should have general utility for examining protein interactions and assembly. In our approach, the nature of the molecular surface buried by the domain contacts, the specificity of the residue-to-residue interactions, and the identity of electrostatic, hydrophobic, and hydrophilic interactions are elucidated. These techniques are applied to the beta barrel domains of Cu, Zn superoxide dismutase (SOD), immunoglobulin Fab, and tomato bushy stunt virus coat protein (TBSV), a plant viral capsid protein. By looking at a set of proteins having different numbers of interacting beta domains, we have been able to see some of the variety and also some of the patterns common to these assembled domains. Strong beta domain interactions (identified by their biochemical integrity) are apparently due to chemical, electrostatic, and shape complementarity of the molecular surfaces buried from interaction with solvent molecules. Although the amount of hydrophobic buried surface area appears to correlate with the strength of the interaction, electrostatic forces appear to be important in both stabilizing and destabilizing specific contacts. In TBSV, analysis of electrostatic interactions may help explain mechanisms of subunit accommodation to different environments, particle expansion, and pathways of assembly. The possible molecular basis for observed differences in the stability and flexibility of the domain complexes is discussed